During FY15, we focussed on the following four subprojects: (i) Over the past 20 years, we have studied many aspects of herpesvirus assembly, focussing mainly on the nucleocapsid. In the past year, we completed a high resolution cryo-EM study of the procapsid of herpes simplex virus type1 (HSV-1) and the early stages of its maturation. The procapsid is made up of 4000 protein subunits of eleven different kinds. As it matures, the procapsid undergoes major changes in structure and composition. In addition to an internal scaffolding protein, assembly is guided by an external scaffolding protein, the triplex, a heterotrimer that coordinates neighboring capsomers. To investigate assembly, we developed a novel isolation procedure for the labile procapsid and collected a large set of cryo-EM data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations, which also yielded new insights into maturation, are to be submitted for publication. (ii) The internal organization of adenovirus. Genome packing in adenovirus has long evaded precise description, since the viral genome condensed by proteins (core) lacks icosahedral order). We combined cry-electron tomography with biophysical analysis to infer the spatial distributions of DNA /protein particles (adenosomes). We found that despite the lack of symmetry the adenosome distribution is not random. The features of their distribution can be explained by modeling the adenosomes as soft spheres, interacting repulsively with each other and with the capsid, producing a minimum outward pressure of 0.06 atm. Although the condensing proteins are connected by DNA in disrupted virion cores, in our models a backbone of DNA linking the adenosomes is not required to explain the experimental results on the confined state. This study was completed and published during FY15 (2). (iii) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system (4). The tailed phages afford an excellent model for herpesvirus capsids, reflecting common evolutionary origins. In FY15, we pursued the coupling of genome packaging to capsid maturation in the cystovirus system. Cystoviruses are double-stranded RNA viruses whose capsids serve as compartments for the replication and transcription of the multiply segmented viral genomes. In FY13, we determined and published a crystal structure for the capsid protein P1, revealing a flattened trapezoid subunit with a novel alfa-helical fold. We also solved the procapsid by cryo-electron microscopy to 0.45 nm resolution. We found that maturation proceeds via two intermediate states and involves some remodeling, besides huge rigid-body rotations (ref). In FY14 and FY15, we have focussed on the binding of the cellular protein YazQ to the outer surface of the mature phi6 capsids. This study is ongoing. (iv) Localizing internal proteins by bubble gram imaging. While many experimental approaches can be used to localize protein domains that are exposed on the surface of a nucleocapsid, few are applicable to proteins that are buried in the interior. We have been exploring the potentiality of radiation damage under cryo-EM conditions for this purpose. Sustained radiation eventually elicits the formation of bubbles of hydrogen gas in proteins that are embedded in DNA. The locations of the bubbles can be detectted in reconstructions calculated from earlier images of the same (undamaged) specimen. Our first success was with PhiKZ, a large and complex virus that infects the pathogenic bacterium Pseudomonas aeruginosa. This was followed up in FY14 with a characterization of the internal structure of bacteriophage T7, viewed as a partially defined model system. In FY15, we investigated capsids of herpes simplex virus (see (i) above)). The procapsid is the earliest precursor and subsequently matures. A-capsids are mature but empty; B-capsids are mature but retain a shrunken internal scaffold; C-capsids are filled with DNA and have ostensibly expelled the scaffolding shell. Among our goals for bubblegram imaging was to determine whether there are any proteins present embedded inDNA inside the C-capsid. We found that procapsids show extensive bubbling in their inner (scaffolding) shell, while the outer shell (capsid) shows only blurring, not bubbling. A-capsids blur but do not bubble. B-capsids bubble internally, confirming that their internal density consists mainly if not exclusively of scaffolding protein. In contrast, C-capsids show small bubbles distributed throughout their interior. A plausible (but not the only) candidate is residual scaffolding protein. This study is now being wrapped up for publication. (v) Papillomavirus maturation. The Papillomaviridae are a family of DNA viruses that inhabit the skin or mucosal tissues of their vertebrate hosts. Unlike other non-enveloped viruses, papillomaviruses are released into the environment through a gradual process called desquamation. During this process, disulfide cross links are formed between neighboring molecules of the major capsid protein, L1. This is thought to stabilize the maturing virion. In FY14, we completed a study in which time-lapse cryo-EM was used to study the maturation of HPV16. Initially, the virion is a loosely connected prolapsed that condenses into the mature papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter, and its pentameric capsomers change in structure (most markedly in their axial region), and the interaction surfaces between adjacent capsomers are consolidated (1). Our current goal is to make a structural characterization of next generation virus-like particles designed for use as vaccines against cervical cancer. (vi) Electron microscopy and tomography of influenza A virus. We have a long-standing interest in the structure, assembly, and membrane fusion-capability of influenza virus. In FY15, we wrote and published a review covering various aspects of electron microscopy that have been applied in this system (3). Biowulf was used in the course of some of these studies.